Thermal cycler optimized for real-time DNA analysis

Editor’s Note: In the following application note, Nextreme details a compact solution for real-time testing in health care, forensics and food safety.

According to industry experts, the global PCR products market is
expected to grow from nearly $5 billion in 2010 to nearly $8 billion by
2015, at an estimated compound annual growth rate (CAGR) of 9.9% from
2010 to 2015. The instruments segment reached $1.7 billion in 2010 and
is expected to grow to nearly $2.7 billion at a compound annual growth
rate (CAGR) of 9.6% from 2010 to 2015.

Much of this growth stems
from the need for thermal cyclers to become more portable, promoting
real-time testing by bringing the testing to the event such as testing
bio-pathogens or DNA identification in the field rather than
transporting samples to the lab.

A new compact thermal cycler reference design for the temperature control of the polymerase chain reaction (PCR) process used for DNA amplification is now available from Nextreme Thermal Solutions.

Polymerase chain reaction (PCR) is a scientific technique that amplifies a single or a few copies of a specific piece of DNA by several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.

Click on image to enlarge.

Figure
1: During DNA replication, each strand
of the original molecule acts as a template for the synthesis of a new,
complementary DNA strand. Source: National Institute of General Medical
Sciences, nigms.nih.gov.

Developed in 1983, PCR has rapidly become one of the most widely used techniques in molecular biology and for good reason: it is a rapid, inexpensive and simple means of producing relatively large quantities of whole or fractional DNA strands copied from minute quantities of source DNA material even when the source DNA is of relatively poor quality.

Applications for PCR include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases.

The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling of the DNA sample based on a predefined series of temperature steps. A thermal cycler is an automated instrument specifically designed for this purpose. A typical device consists of a metal block with holes where plastic vials holding the PCR reaction mixtures are inserted. The instrument has an integrated heating/cooling unit that is used to systematically raise and lower the temperature of the block. Thermoelectric coolers (TECs) are used for a large number of these systems.

Thermoelectric cooling makes use of the Peltier effect to create a heat flux between the junctions of two different types of materials. A Peltier cooler, heater, or thermoelectric heat pump is a solid-state heat pump that transfers heat from one side of the device to the other side against a temperature gradient (from cold to hot). To do this, electrical energy is required. The electrical energy used for the pumping is converted into additional heat that must be removed from the system (much like the heat from a home air-conditioner unit).

The most basic representation of a thermoelectric cooling device is a cooling curve (Figure 2). The cooling curve represents the ?T (or temperature difference between the cold and hot sides of the TEC) as a function of the input electrical current to the TEC. There is a different curve for each unique amount of heat being pumped. Figure 2 shows three different examples. As the heat pumped (Q) increases, the amount the TEC can cool is reduced. For all cases, the maximum drive current for the module, Imax, provides the most cooling achievable from that TEC under the given load conditions.

Click on image to enlarge.

Figure 2: The cooling curve of a thermoelectric cooler at three different heat loads (10%, 30% and 50% of the Qmax or maximum allowable heat load. TEC

Thin-film thermoelectric coolers (eTECs) operate in the same manner as conventional ones but offer several key advantages that are particularly well suited for PCR. These advantages are:

Figure 3: Size comparison of a 4 W Nextreme eTEC to a conventional TEC with the same heat pumping capacity.

In the Nextreme thin-film reference design, Figure 4, the thermal subsystem consists of the sample cartridge holder, support platform, thin-film thermoelectric module with integrated heat spreader interface, and heat sink (usually with fan). In conventional PCR systems intended for laboratory usage, multiple samples (e.g., 96 or more) are cycled together using a single large sample side heat spreader and a bulk thermoelectric device. However, the current market shift towards doctor's office or patient side usage systems only need to handle one to four samples at a time with potentially different protocol thermal requirements for each sample. The microscopic size of the Nextreme eTEC enables different temperatures in different parts of the block – something that cannot be achieved with conventional technology.

Thermal cycle times in PCR thermal cyclers are determined both by the dwell times during the denaturation, annealing, and extension phases and the thermal transition time between these phases. Thermal cycle time is minimized and throughput maximized by minimizing the transition time between the phases. Conventional PCR systems use large individual sample volumes (e.g.; 100 µL) and temperature transitions at 1 - 5 °C/s. However, while most PCR protocols are performed at the 25 µL to 50 µL scale, sample volume as low as 5 µL have also been shown to be successful using Nextreme thin-film modules.

The technology can enable a new generation of compact thermal cyclers for equipment manufacturers that lower the barriers-to-entry and increase opportunities for differentiation.

The high heat pumping capacity per unit area of the thin-film modules, along with their inherent rapid response, enables extremely rapid temperature transitions in the sample. For optimized designs, temperature transition rates in the range of 20°C/s to 30°C/s are feasible for currently used sample volumes. For smaller sample volumes, even faster temperature transitions rates are possible.

Nextreme has conducted rigorous reliability tests on the eTEC family of thermoelectric modules. The devices have surpassed baseline tests in mechanical shock, thermal storage and power cycling. In a recent power cycling experiment in particular, modules were subjected to over 500,000 power cycles with little change in AC resistance, a key measure of reliability and performance.

This result is particularly important as the testing was conducted at an electrical current that far exceeds normal operating conditions, furthering indicating stable device performance over a large number of cycles. In all cases, the results strongly indicate modules are highly reliable for use in PCR thermal cycling applications.

Nextreme offers several thermoelectric coolers that are designed for thermal cycling applications. Nextreme recommends the use of its thermal modeling, design and engineering services to deliver fully-optimized PCR thermal cycling solutions.

About the authorKarl von Gunten is the Director of Marketing for Nextreme. Prior to joining Nextreme, von Gunten was director of business
development, managing supervisor, at Gibbs & Soell Public Relations
and vice president at Brodeur Worldwide, where he oversaw the regional
office and managed PR efforts for industry leaders such as IBM, Nortel,
Acterna, and Internet Security Systems. He serves as an adjunct
professor for Duke University Pratt School of Engineering and holds a
B.A. in Physics from Wittenberg University.